DNA trapping electrophoresis.
Attempts to improve the size separation of single-stranded DNA in polyacrylamide gels by field-inversion gel electrophoresis (FIGE) have met with limited success. Here we show that attaching a neutral globular protein, streptavidin, to one end of a single-stranded DNA molecule profoundly alters the DNA mobility pattern and increases the band separation manyfold within a size range controlled by voltage and pulse cycle. In constant field, short modified fragments are only slightly retarded but long molecules are retarded dramatically above a 'threshold size' of 0.6 kilobases at 60 V per cm. At this voltage, molecules above a 1.2-kilobase 'cut-off' do not enter the gel. Both the threshold and the cut-off sizes decrease as the voltage increases. In FIGE, the longer the reverse pulse, the larger the modified fragments that enter the gel. We interpret these results as the trapping by the gel matrix of the protein attached to the DNA. The probability of release then depends on the balance between the electric field and thermal motion: the larger the DNA and the higher the voltage, the harder it is to release.
Ulanovsky
L
L
Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
Drouin
G
G
Gilbert
W
W
D004247:DNA / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005559:Formamides; D007202:Indicators and Reagents; D008970:Molecular Weight
D004277:DNA, Single-Stranded; D005559:Formamides; D007202:Indicators and Reagents; C031066:formamide; D004247:DNA